Negative information for motif discovery.

We discuss a method of combining genome-wide transcription factor binding data, gene expression data, and genome sequence data for the purpose of motif discovery in S. cerevisiae. Within the word-counting algorithmic approach to motif discovery, we present a method of incorporating information from negative intergenic regions where a transcription factor is thought not to bind, and a statistical significance measure which account for intergenic regions of different lengths. Our results demonstrate that our method performs slightly better than other motif discovery algorithms. Finally, we present significant potential new motifs discovered by the algorithm.

Selectivity of F8-actinomycin D for RNA:DNA hybrids and its anti-leukemia activity.

Although many compounds have been found that bind to DNA in various ways and exhibit various biological activities, few compounds that specifically bind to RNA or RNA:DNA hybrids are known, even though such compounds are expected to have important biological properties. For example, one characteristic function of the retroviruses, which is generally not found in eukaryotic cells, is the production of an RNA:DNA hybrid in the viral replication phase. If an agent is designed to bind only to an RNA:DNA hybrid, and not to DNA or to RNA, such an agent might be able to inhibit specifically the RNase H activity of retroviral reverse transcriptase, and therefore suppress viral replication. Actinomycin D is known to bind to double-stranded DNA, but not to RNA, because steric hindrance between the 2-amino group of the phenoxazone ring and the 2'-hydroxyl group of RNA prevents intercalation of the compound. However, if the > C-H moiety at the 8-position of the phenoxazone ring is replaced by a > C-F, a possible hydrogen-bond acceptor, this analogue (8-fluoro-actinomycin D, F8AMD) might be able to bind intercalatively to an RNA:DNA hybrid by forming an additional hydrogen bond between F8 and the 2'-hydroxyl group of the guanosine ribose. To test this hypothesis, the crystal structure of d(GAAGCTTC)2-F8AMD has been determined at 3.0 A resolution. Based on this crystal structure, a model in which F8AMD binds into the hybrid r(GAAGCUUC):d(GAAGCTTC) has been built using molecular mechanics and dynamic methods. These structural studies indicate that F8AMD binds intercalatively to a B-form double-stranded DNA whereas the drug intercalates into an RNA:DNA hybrid taking an A-form conformation. In the RNA:DNA hybrid complex, the F8 atom is located so as to be able to interact to an O2' hydroxyl group with either an O-H...F hydrogen bond or H+...F- electrostatic interaction. This interaction might stabilize the F8AMD molecule in the RNA:DNA hybrid. A binding study indicates that both actinomycin D (AMD) and F8AMD bind intercalatively not only to double-stranded DNAs, but also to RNA:DNA hybrids. Although the overall binding capacity of F8AMD (k = 4.5 x 10(5) M-1) is reduced slightly in comparison with AMD itself (k = 1.8 x 10(6) M-1), F8AMD tends to bind relatively more favorably than AMD to the RNA:DNA hybrids. The drugs' effects on RNA synthesis in HeLa cells indicates that the binding capacities of AMD and F8AMD correlates strongly to their RNA synthesis inhibitory activities. F8AMD required a concentration of 78 nM to inhibit RNA polymerase activity in HeLa cells by 50%, whereas AMD reached the same inhibitory level at 30 nM. Surprisingly, F8AMD exhibits unique selectivity against leukemia cells as does another C8-derivatized AMD analogue, N8AMD. F8AMD inhibits 50% of leukemia cell growth at less than 1.0 nM whereas 10- to 130-fold-higher drug concentrations are required to inhibit the growth of other tumor cell lines by 50%. The GI50 value of F8AMD for leukemia cells is the lowest among the GI50 values for all other AMD derivatives tested. By contrast, AMD is quite potent and kills most cells at less than 50 nM concentration, but it does not show any selectivity for certain cell lines. This indicates that AMD should have very limited use as an antitumor agent. It is difficult to rationalize why F8AMD and N8AMD show such strong selectivity against leukemia cells. However, this study and our previous study (J. Am. Chem. Soc. 1994, 116, 7971) indicated that F8AMD and N8AMD tended to bind more favorably to RNA:DNA hybrids. Thus, the unique antileukemia selectivity shown by F8AMD and N8AMD might be used by the agents binding to RNA:DNA hybrids rather than to double-stranded DNA.

Physical and biological characteristics of the antitumor drug actinomycin D analogues derivatized at N-methyl-L-valine residues.

The crystal structure of the DNA-actinomycin D (AMD) complex and a simple molecular modeling study indicated that AMD analogues derivatized at N-methyl-L-valine residues (fifth amino acid residue in the cyclic depsipeptide of AMD) could bind to DNA as strongly as the parent AMD. The analogues in which N-methyl-L-valine residues were replaced with L- and D-forms of N-methylvalines, N-methylthreonines, N-methylphenylalanies, N-methyltyrosines, and N-methyl-O-methyltyrosines have been totally synthesized. The characteristics of binding of the analogues to various DNAs including DNA-1 [d(TATATATGCATATATA)], DNA-2 [d(TATATACGCGTATATA)], DNA-3 [d(ATATATAGCTATATAT)], and DNA-4 [d(ATATATGGCCATATAT)] have been examined by using visible absorption spectrum methods. The association constants calculated from the absorption spectra indicate that the modifications of the N-methyl-L-valine residues in the AMD molecule do affect the DNA binding characteristics of the analogues. The L-aromatic analogues bind slightly better than the L-aliphatic analogues except for binding to DNA-1 (-TGCA-), whereas the D-aliphatic analogues bind consistently better than the D-aromatic analogues. In the L-form analogues, the L-Tyr analogue has the highest overall association constant, whereas the D-Val analogue has the highest association constant among the D-form analogues. In spite of substitution of bulky aromatic groups, the D-aromatic analogues bind to the DNA-1 quite well. However, D-aromatic analogues have significantly reduced their binding capacities to the other DNAs, indicating that the substitution of the D-aromatic residues creates a unique four-base sequence preference (-TGCA-). The RNA polymerase inhibitory activities of the AMD analogues in vivo have been examined using human cells (HeLa). All AMD analogues except for the L-Thr analogues severely inhibit RNA synthesis at relatively low drug concentrations. The D-Val, L-OMT, L-Phe, and D-Phe analogues inhibit RNA synthesis more strongly than the natural antibiotic (AMD itself).

Conversion of Lewis lung carcinoma into ascitic form.

Lewis lung carcinoma (3LL), which has been most frequently used for various investigations of cancer, was converted to an ascitic form designated 3LL-SA. Ascites tumor cells showed a mixed population of single cells and about 10% islands which were made up of 2 to about 50 cells. Transplantability was 100%. The biological and metastatic capacities were preserved in this line as shown by its ability to form hemorrhagic and necrotic tumors, and by its multiple lung metastases. Moreover, histological and ultrastructural characteristics of this tumor line were identical to those of the parental 3LL. Thus, it can be seen that 3LL-SA will be a useful tool for the study of cancer.

Immunoelectron microscopic observations on the localization of fibronectin in normal human lung.

The localization of fibronectin was examined in normal human lung using immunoelectron microscopy. Fibronectin staining was present not only in the endothelial, alveolar epithelial and bronchial epithelial basal laminae and associated with interstitial collagen fibrils and elastic fibers, but also in basal laminae of fibroblasts and smooth muscle cells. There was a periodicity in the staining of fibronectin on collagen fibrils. Reaction products against fibronectin were present in intracellular organelle including cisternae of smooth endoplasmic reticulum of endothelial cells and those of rough endoplasmic reticulum of fibroblasts. No other cells contained reaction products. The localization of fibronectin was compared with that of ruthenium red staining in normal human lung. The localization of fibronectin was consistent with that of proteoglycan.

Pattern of fibronectin distribution in human lung cancer.

The pattern of fibronectin (FN) distribution in human lung cancer was studied by indirect immunofluorescent staining, and by the peroxidase-antiperoxidase method in a total of 60 surgical specimens. They comprised 8 small cell carcinomas, 4 large cell carcinomas, 19 squamous cell carcinomas, 28 adenocarcinomas, and 1 adenoid cystic carcinoma. Of the 60 specimens 13 were FN-positive. They included 4 large cell carcinomas, 4 small cell carcinomas, 3 poorly differentiated squamous cell carcinomas, and 2 poorly differentiated and 1 moderately differentiated adenocarcinomas. On the other hand, none of the well differentiated carcinomas was FN-positive around tumor cells. Our data suggest that undifferentiated, or poorly differentiated carcinomas of the lung tend to be FN-positive.

Abnormal cilia in the small airways of mice induced by bleomycin.

The frequency and types of abnormal cilia in the small airways before and after parenteral administration of bleomycin were examined by transmission electron microscopy. Bleomycin was injected subcutaneously at a dose of 40 mg/kg twice weekly for 6 weeks. Bronchioles less than 0.1 mm in diameter from animals sacrificed 1 day after a single dose, 1 day after three doses, 3 days after 8 doses, and 13 days after 12 doses were selected for evaluation. Although ciliated cells proper did not show pronounced alterations, abnormal cilia such as swollen cilia, compound cilia, and cilia with abnormal axonemes frequently occurred. Repeated injections did not necessarily induce much more abnormal cilia than one dose. The results suggested that ciliated cells remain undamaged and that normal cilia can be restored. The specific type of abnormal cilia induced by bleomycin was not identified.

Ultrastructure of the Lewis lung carcinoma.

ELectron microscopic observations were made on the Lewis lung carcinoma, which has frequently been used in various experiments. Although the tumor cells had desmosomes, interdigitation, microvilli and basement membranes which were of epithelial nature, they did not show either squamous or adenomatous differentiation. On the basis of microscopic and ultrastructural findings, this tumor falls into the category of large cell carcinomas. At the periphery of the tumor, sinusoidal clefts between the strands of tumor cells contained many red blood cells without fibrin. While perfect blood vessels were observed towards the center of the tumor, large necrotic areas were observed. Dilated large vessels possessed some endothelial fenestrations. Light endothelial cells were also observed, which were penetrated by red blood cells. Interstitial components were very scant and collagen fibres were frequently observed to be dissolved.

Electron microscopic observations on pulmonary connective tissue stained by Ruthenium Red.

Ultrastructural studies on human lung were performed with special attention to the interstitial acid mucopolysaccharides by Ruthenium Red staining and several enzyme digestion tests with Streptomyces hyaluronidase, chondroitinase ABC, chondroitinase AC, heparinase, trypsin and collagenase. Periodic lateral granules on the major cross bands of collagen fibrils and amorphous coats on them became visible by Ruthenium Red staining. The surface of elastic fibres, associated microfibrils, and some fine fibrils 10-20 nm in diameter were stained. Ruthenium Red also stained the surface of fibroblast and smooth muscle cells, basement membrane and filamentous long segments. In the interstructural space, granular substances 10-80 nm in diameter and fine filaments 3--4 nm thick, which formed a fine reticular network, were clearly observed. They were not visible on the usual thin section. The granular substances were located on the cross points of the fine filaments. They spread continuously and connected with each of the cells and extracellular structures in the pulmonary interstitium. The results of the enzyme digestion tests on the Ruthenium Red-positive material are discussed.

Cytoplasmic microtubules of rat ascites hepatoma cells.

Cytoplasmic microtubules of 4 strains of rat ascites hepatoma cells including YS, AH 66F, AH 130 and AH 100B were investigated electron microscopically. Microtubules were clearly demonstrated when the cells were fixed at 20 degrees C or 37 degrees C and stained by tannic acid. Morphology, localization and volume density (AH 130 greater than AH 66F greater than YS greater than AH 100B) of microtubules were examined comparatively in these 4 strains and correlation between microtubules and cell deformability was discussed.

A biochemical study on bronchopulmonary lavage fluid from a case of pulmonary alveolar proteinosis.

A 44-year-old male was hospitalized, because of the presence of abnormal shadows on chest x-ray film. Diagnosis of pulmonary alveolar proteinosis was established by examination of a specimen obtained by transbronchial biopsy. Therapeutic bronchopulmonary lavage was performed twice. As a result, there was an improvement of radiological findings on chest x-ray film. Then a biochemical study was carried out with pooled lavage fluids. Cellular debris were removed by centrifugation. Lipids were extracted with a 2:1 mixture of chloroform and methanol. Individual phospholipids were identified by column chromatography and thin layer chromatography. Fatty acids were identified by gas chromatography. It was found that phosphatidyl choline (lecithine) was the major component of phospholipids. On the other hand, myristic acid of the molecule of phosphatidyl choline was found to constitute 19.6% of the total fatty acids. This patient lives a normal life as of Dec. 1980.

Lysosomal accumulation of gallium-67 in Morris hepatoma-7316A and Shionogi mammary carcinoma-115.

Intracellular localization of gallium-67 was investigated in Morris hepatoma-7316A and Shionogi mammary carcinoma-115 cells by the cell fractionation method 48 hr after an intraperitoneal injection of the nuclide. When lysosomes were purified from both tumors by discontinuous sucrose density gradient centrifugation, they had a strikingly high relative specific activity of the nuclide. From these results it was confirmed that gallium-67 is concentrated most specifically in the lysosomes of both tumor cells, which consist chiefly of phagolysosomes and can engulf only limited amount of foreign materials such as Triton and gallium-67.

Electron microscopy of C-particles and small vesicles in a transplantable rat ascites hepatoma-66F.

Transplantable rat ascites hepatoma AH-66F was examined by an electron microscope. C-particles were numerously observed in the intercellular spaces and on the cell surface. The size of these particles varied from 90 to 200 nm in diameter. Small vesicles were also observed. These vesicles ranged from 40 to 50 nm in diameter. Most of them were located near the group of C-particles. Occasionally, small vesicles were observed in the form of a rosette around the C-particles.